Biophosphonate compounds and methods for bone resorption diseases, cancer, bone pain, immune disorders, and infectious diseases

ABSTRACT

Bisphosphonate compounds and related methods of making and using are disclosed, including pyridinium-1-yl, quinolinium-1-yl, and related compounds. The activity of compounds is disclosed in the context of functional assays such as  Leishmania major  farnesyl diphosphate synthase (FPPS) inhibition,  Dictyostelium discoideum  growth inhibition, human gamma delta T cell activation, and bone resorption. The applicability of bisphosphonate compounds in the context of parasitic infections, for example against trypanosomes, is disclosed. Further potential applications of the invention are disclosed regarding the treatment of one or more conditions such as bone resorption disorders, cancer, bone pain, infectious diseases, and in immunotherapy.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application 60/617,108 by Sanders et al., filed Oct. 8, 2004, which is incorporated by reference in entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made, at least in part, with government support under Grant Nos. GM50694 and GM65307 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Nitrogen-containing bisphosphonates such as pamidronate (Aredia®) C1; alendronate (Fosamax®) C2; risedronate (Actonel®) C3; and zoledronate (Zometa®) C4; shown in their zwitterionic forms in FIG. 1, represent an important class of drugs, currently used to treat osteoporosis, Paget's disease and hypercalcemia due to malignancy. See references 1-4. These compounds function primarily by inhibiting the enzyme farnesyl diphosphate synthase (FPPS) (references 5-12) resulting in decreased levels of protein prenylation in osteoclasts (references 13-15). Certain bisphosphonates have also been found to have anti-parasitic activity (references 16-25) and have been found to stimulate human γδ T cells (references 26-30); there is currently interest in their use as vaccines for a variety of B cell malignancies (reference 31).

Differences in substituents, however, can strongly influence the pharmacologic properties of such compounds (Green, 2001). Structural differences may also be significant in the potential expansion of therapies. For example, Bonefos (clodronate) is a bisphosphonate indicated for the treatment of tumor-induced osteolysis and hypercalcemia. It has been reported to increase survival and reduce the risk of bone metastasis in women with stage II/III breast cancer. This is noteworthy as approximately 70% of women who develop recurrence of breast cancer will experience bone metastasis, and breast cancer remains the leading cause of death among women aged 40 to 55 years.

For even second generation bisphosphonates, it is recognized that small changes of structure can lead to marked improvements in activity or function, for example in the inhibition of osteoclastic resorption potency (Widler et al., 2002). Therefore, there is great interest in the further development of alternative bisphosphonate compounds and the exploration of methods of use such as clinical applications.

SUMMARY OF THE INVENTION

The present invention surprisingly provides the first report of the synthesis and testing of a series of pyridinium-1-yl and related bisphosphonates. Bisphosphonate compounds of the invention can demonstrate activity in one or more contexts, including a farnesyl diphosphate synthase (FPPS) assay, a D. discoideum growth inhibition assay, a T cell activation assay, a bone resorption assay, the treatment of infectious disease, the treatment of a bone resorption clinical disorder, an immunotherapeutic treatment, the treatment of cancer, and the treatment of bone pain.

The invention broadly provides bisphosphonate compounds and related methods of making and using. The invention specifically provides compounds with an N-linkage including pyridinium-1-yl, quinolinium-1-yl, and related bisphosphonate compounds.

The following abbreviations are applicable. FPPS, farnesyl diphosphate synthase; pIC₅₀/pEC₅₀, negative log of IC₅₀ and EC₅₀, respectively, where IC₅₀ and EC₅₀ are the concentrations that produce half-maximal inhibition or activation, respectively; L. major, Leishmania major, D. discoideum, Dictyostelium discoideum; γδ T cells, gammadelta T cells. Compounds are optionally designated by a number or in some cases a number preceded by a letter to help distinguish a compound designation from a cardinal number, e.g. C1 is compound 1.

The following definitions are applicable.

Alkyl groups include straight-chain, branched and cyclic alkyl groups. Alkyl groups include those having from 1 to 20 carbon atoms. Alkyl groups include small alkyl groups having 1 to 3 carbon atoms. Alkyl groups include medium length alkyl groups having from 4-10 carbon atoms. Alkyl groups include long alkyl groups having more than 10 carbon atoms, particularly those having 10-20 carbon atoms. Cyclic alkyl groups include those having one or more rings. Cyclic alkyl groups include those having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring and particularly those having a 3-, 4-, 5-, 6-, or 7-member ring. The carbon rings in cyclic alkyl groups can also carry alkyl groups. Cyclic alkyl groups can include bicyclic and tricyclic alkyl groups. Alkyl groups optionally include substituted alkyl groups. Substituted alkyl groups include among others those which are substituted with aryl groups, which in turn can be optionally substituted. Specific alkyl groups include methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, n-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl, and cyclohexyl groups, all of which are optionally substituted.

Alkenyl groups include straight-chain, branched and cyclic alkenyl groups. Alkenyl groups include those having 1, 2 or more double bonds and those in which two or more of the double bonds are conjugated double bonds. Alkenyl groups include those having from 2 to 20 carbon atoms. Alkenyl groups include small alkyl groups having 2 to 3 carbon atoms. Alkenyl groups include medium length alkenyl groups having from 4-10 carbon atoms. Alkenyl groups include long alkenyl groups having more than 10 carbon atoms, particularly those having 10-20 carbon atoms. Cyclic alkenyl groups include those having one or more rings. Cyclic alkenyl groups include those in which a double bond is in the ring or in an alkenyl group attached to a ring. Cyclic alkenyl groups include those having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring and particularly those having a 3-, 4-, 5-, 6- or 7-member ring. The carbon rings in cyclic alkenyl groups can also carry alkyl groups. Cyclic alkenyl groups can include bicyclic and tricyclic alkyl groups. Alkenyl groups are optionally substituted. Substituted alkenyl groups include among others those which are substituted with alkyl or aryl groups, which groups in turn can be optionally substituted. Specific alkenyl groups include ethenyl, prop-1-enyl, prop-2-enyl, cycloprop-1-enyl, but-1-enyl, but-2-enyl, cyclobut-1-enyl, cyclobut-2-enyl, pent-1-enyl, pent-2-enyl, branched pentenyl, cyclopent-1-enyl, hex-1-enyl, branched hexenyl, cyclohexenyl, all of which are optionally substituted.

Aryl groups include groups having one or more 5- or 6-member aromatic or heteroaromatic rings. Aryl groups can contain one or more fused aromatic rings. Heteroaromatic rings can include one or more N, O, or S atoms in the ring. Heteroaromatic rings can include those with one, two or three N, those with one or two O, and those with one or two S. Aryl groups are optionally substituted. Substituted aryl groups include among others those which are substituted with alkyl or alkenyl groups, which groups in turn can be optionally substituted. Specific aryl groups include phenyl groups, biphenyl groups, pyridinyl groups, and naphthyl groups, all of which are optionally substituted.

Arylalkyl groups are alkyl groups substituted with one or more aryl groups wherein the alkyl groups optionally carry additional substituents and the aryl groups are optionally substituted. Specific alkylaryl groups are phenyl-substituted alkyl groups, e.g., phenylmethyl groups.

Alkylaryl groups are aryl groups substituted with one or more alkyl groups wherein the alkyl groups optionally carry additional substituents and the aryl groups are optionally substituted. Specific alkylaryl groups are alkyl-substituted phenyl groups such as methylphenyl.

The rings that may be formed from two or more of R¹-R⁵ together can be optionally substituted cycloalkyl groups, optionally substituted cycloalkenyl groups or aromatic groups. The rings may contain 3, 4, 5, 6, 7 or more carbons. The rings may be heteroaromatic in which one, two or three carbons in the aromatic ring are replaced with N, O or S. The rings may be heteroalkyl or heteroalkenyl, in which one or more CH₂ groups in the ring are replaced with O, N, NH, or S.

Optional substitution of any alkyl, alkenyl and aryl groups includes substitution with one or more of the following substituents: halogens, —CN, —COOR, —OR, —COR, —OCOOR, —CON(R)₂, —OCON(R)₂, —N(R)₂, —NO₂, —SR, —SO₂R, —SO₂N(R)₂ or —SOR groups. Optional substitution of alkyl groups includes substitution with one or more alkenyl groups, aryl groups or both, wherein the alkenyl groups or aryl groups are optionally substituted. Optional substitution of alkenyl groups includes substitution with one or more alkyl groups, aryl groups, or both, wherein the alkyl groups or aryl groups are optionally substituted. Optional substitution of aryl groups includes substitution of the aryl ring with one or more alkyl groups, alkenyl groups, or both, wherein the alkyl groups or alkenyl groups are optionally substituted.

Optional substituents for alkyl, alkenyl and aryl groups include among others:

—COOR where R is a hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which are optionally substituted;

—COR where Ris a hydrogen, or an alkyl group or an aryl groups and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted;

—CON(R)₂ where each R, independently of each other R, is a hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted; R and R can form a ring which may contain one or more double bonds;

—OCON(R)₂ where each R, independently of each other R,is a hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted; R and R can form a ring which may contain one or more double bonds;

—N(R)₂ where each R, independently of each other R, is a hydrogen, or an alkyl group, acyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl or acetyl groups all of which are optionally substituted; or R and R can form a ring which may contain one or more double bonds.

—SR, —SO₂R, or —SOR where R is an alkyl group or an aryl groups and more specifically where R is methyl, ethyl, propyl, butyl, phenyl groups all of which are optionally substituted; for —SR, R can be hydrogen;

—OCOOR where R is an alkyl group or an aryl groups;

—SO₂N(R)₂ where R is a hydrogen, an alkyl group, or an aryl group and R and R can form a ring;

—OR where R═H, alkyl, aryl, or acyl; for example, R can be an acyl yielding —OCOR* where R* is a hydrogen or an alkyl group or an aryl group and more specifically where R* is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted;

Specific substituted alkyl groups include haloalkyl groups, particularly trihalomethyl groups and specifically trifluoromethyl groups. Specific substituted aryl groups include mono-, di-, tri, tetra- and pentahalo-substituted phenyl groups; mono-, di-, tri-, tetra-, penta-, hexa-, and hepta-halo-substituted naphthalene groups; 3- or 4-halo-substituted phenyl groups, 3- or 4-alkyl-substituted phenyl groups, 3- or 4-alkoxy-substituted phenyl groups, 3- or 4-RCO-substituted phenyl, 5- or 6-halo-substituted naphthalene groups. More specifically, substituted aryl groups include acetylphenyl groups, particularly 4-acetylphenyl groups; fluorophenyl groups, particularly 3-fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups, particularly 3-chlorophenyl and 4-chlorophenyl groups; methylphenyl groups, particularly 4-methylphenyl groups, and methoxyphenyl groups, particularly 4-methoxyphenyl groups.

Pharmaceutically acceptable salts comprise pharmaceutically-acceptable anions and/or cations. Pharmaceutically-acceptable cations include among others, alkali metal cations (e.g., Li⁺, Na⁺, K⁺), alkaline earth metal cations (e.g., Ca²⁺, Mg²⁺), non-toxic heavy metal cations and ammonium (NH₄ ⁺) and substituted ammonium (N(R′)₄ ⁺, where R′ is hydrogen, alkyl, or substituted alkyl, i.e., including, methyl, ethyl, or hydroxyethyl, specifically, trimethyl ammonium, triethyl ammonium, and triethanol ammonium cations). Pharmaceutically-acceptable anions include among other halides (e.g., Cl⁻, Br⁻), sulfate, acetates (e.g., acetate, trifluoroacetate), ascorbates, aspartates, benzoates, citrates, and lactate.

Compounds of the invention can have prodrug forms. Prodrugs of the compounds of the invention are useful in the methods of this invention. Any compound that will be converted in vivo to provide a biologically, pharmaceutically or therapeutically active form of a compound of the invention is a prodrug. Various examples and forms of prodrugs are well known in the art. Examples of prodrugs are found, inter alia, in Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985), Methods in Enzymology, Vol. 42, at pp. 309-396, edited by K. Widder, et. al. (Academic Press, 1985); A Textbook of Drug Design and Development, edited by Krosgaard-Larsen and H. Bundgaard, Chapter 5, “Design and Application of Prodrugs,” by H. Bundgaard, at pp. 113-191, 1991); H. Bundgaard, Advanced Drug Delivery Reviews, Vol. 8, p.1-38 (1992); H. Bundgaard, et al., Journal of Pharmaceutical Sciences, Vol. 77, p. 285 (1988); and Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392).

The invention provides compounds having the formula CA1:

(see also FIG. 9)

or a pharmaceutically acceptable salt, ester or hydrate thereof; wherein:

X is H, —OH, or a halogen;

n is 1, 2, or 3;

R¹-R⁵, independently of one another and other R groups, are selected from the group consisting of a hydrogen, a halogen, a —CN, —OR, —COOR, , —OCOOR, —COR, —CON(R)₂, —OCON(R)₂, —N(R)₂, —NO₂, —SR, —SO₂R, —SO₂N(R)₂ or —SOR group, an optionally substituted alkyl group, an optionally substituted alkenyl group, and an optionally substituted aryl group, where each R, independent of any other R in any listed group, is selected from H, an optionally substituted alkyl group, an optionally substituted aryl group, and an optionally substituted acyl group;

two or more of R¹-R⁵ can together form one or more rings which may contain one or more double bonds or which may be aromatic;

R⁶ and R⁷, independently of each other and other R⁶ and R⁷ in the compound, are selected from the group consisting of a hydrogen, a halogen, a —N(R)₂, or —SR group, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkoxy group, and an optionally substituted aryl group, where each R, independent of any other R in any listed group, is selected from H, an optionally substituted alkyl group and an optionally substituted aryl group; and

wherein R⁶ and R⁷ can together form a ring which may contain one or more double bonds.

In specific embodiments, the invention relates to compounds having the above formula where X is OH.

In other specific embodiments, the invention relates to compounds having the above formula where X is H.

In other specific embodiments, compounds of the invention are those of formula CA1, with the exception of the compound of formula CA1 where X is H, n is 1 and all of R¹-R⁷ are hydrogens.

In other specific embodiments, the invention relates to compounds having the above formula wherein n is 1.

In other specific embodiments, the invention relates to compounds having the above formula where X is OH and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein one or both of R⁶ and R⁷ are hydrogens.

In other specific embodiments, the invention relates to compounds having the above formula wherein both of R⁶ and R⁷ are hydrogens.

In other specific embodiments, the invention relates to compounds having the above formula wherein both of R⁶ and R⁷ are hydrogens and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein both of R⁶ and R⁷ are hydrogens, n is 1 and X is OH.

In other specific embodiments, the invention relates to compounds having the above formula wherein both of R⁶ and R⁷ are hydrogens, n is 1 and X is H.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹-R⁵ are all hydrogens.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹-R⁵ are all hydrogens, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹-R⁵ are all hydrogens, X is H and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogen, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogen, X is H and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogen and one or more of R², R³ or R⁴ is a halogen.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴is a halogen, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴ is a halogen, X is H and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, and one or more of R², R³ or R⁴ is an optionally substituted alkyl group, particularly a small alkyl group and more particularly a methyl group.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴is an optionally substituted alkyl group, particularly a small alkyl group, X is OH and n is 1. Specific compounds of this invention are those as above in which R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴ is a methyl group, X is OH and n is 1. Specific compounds of this invention are those as above in which R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴ is a trifluoromethyl group, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴is an optionally substituted alkyl group, particularly a small alkyl group, X is H and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, and one or more of R², R³ or R⁴ is an optionally substituted alkoxy group. A specific alkoxy group is a methoxy group. Specific compounds of this invention are those as in the formula above in which R² or R³is a methoxy group.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, and one or more of R², R³ or R⁴ is an optionally substituted alkoxy group, X is OH and n is 1. Specific compounds of the invention are those as in the formula above wherein R¹ and R⁵ are both hydrogens, R² or R³ is a methoxy group, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, and one or more of R², R³ or R⁴ is an optionally substituted alkoxy group, X is H and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, and one or more of R², R³ or R⁴ is an optionally substituted phenyl group.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴is an optionally substituted phenyl group, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴is an optionally substituted phenyl group, X is H and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, and one or more of R², R³ or R⁴ is an alkyl-substituted phenyl group. Specific alkyl groups are methyl, ethyl and n-propyl groups. Specific compounds of this invention are those as above in which R² or R³is a 3- or 4-alkylphenyl group. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-methylphenyl group. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-ethylphenyl group. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-n-butylphenyl group.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴ is an alkyl-substituted phenyl group, X is OH and n is 1. Specific alkyl groups are methyl, ethyl and n-propyl groups. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-methylphenyl group, X is OH and n is 1. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-ethylphenyl group, X is OH and n is 1. Specific compounds of this invention are those as above in which R² or R³is a 3- or 4-n-propylphenyl group, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴ is an alkyl-substituted phenyl group, X is H and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, and one or more of R², R³ or R⁴ is a halo-substituted phenyl group. Specific halogens are fluorine, chlorine and bromine. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-halophenyl group or a 3-, 4-dihalophenyl group. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-fluorophenyl group. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-chlorophenyl group. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-bromophenyl group. Specific compounds of this invention are those as above in which R² or R³is a 3-bromo- 4-fluorophenyl group or a 3-chloro-4-fluorophenyl group.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴is a halo-substituted phenyl group, X is OH and n is 1. Specific halogens are fluorine, chlorine, and bromine. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-fluorophenyl group, X is OH and n is 1. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-chlorophenyl group, X is OH and n is 1. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-bromophenyl group, X is OH and n is 1. Specific compounds of this invention are those as above in which R² or R³ is a 3-bromo- 4-fluorophenyl group or a 3-chloro-4-fluorophenyl group, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴is a halo-substituted phenyl group, X is H and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, and one or more of R², R³ or R⁴ is a hydroxy-substituted phenyl group which may be in the form of a phenoxy anion or salt thereof. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-hydroxyphenyl group. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-hydroxyphenyl group. Specific compounds of this invention are those in which R² or R³is a 3- or 4-oxyphenyl anion or a salt thereof. Salts of the oxyphenyl anion include Na⁺, K⁺, and other pharmaceutically acceptable salts containing pharmaceutically acceptable cations.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴is a hydroxy-substituted phenyl group, which may be in the form of a phenoxy anion or salt thereof, X is OH and n is 1. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4- hydroxyphenyl group, X is OH and n is 1. Specific compounds of this invention are those in which R² or R³ is a 3- or 4-oxyphenyl anion or a salt thereof, X is OH, and n is 1. Salts of the oxyphenyl anion include Na⁺, K⁺, and other pharmaceutically acceptable salts containing pharmaceutically acceptable cations.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, and one or more of R², R³ or R⁴ is an alkoxy-substituted phenyl group. A specific alkoxy group is a methoxy group. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-methoxy phenyl group.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴ is an alkoxy substituted phenyl group, X is OH and n is 1. A specific alkoxy group is a methoxy group. Specific compounds of this invention are those as above in which R² or R³ is a 3- or 4-methoxy phenyl group, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, and one or more of R², R³ or R⁴ is an optionally substituted arylalkyl group. A specific arylalkyl group is a phenylmethyl group, particularly the compound as above wherein R² or R³is a phenylmethyl group.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴ is an optionally substituted arylalkyl group, X is OH and n is 1. A specific compound of this invention is one in which R¹ and R⁵are both hydrogens, R² or R³is a phenylmethyl group, X is OH and n is 1.

In other specific embodiments, the invention relates to compounds having the above formula wherein R¹ and R⁵ are both hydrogens, one or more of R², R³ or R⁴ is an optionally substituted arylalkyl group, X is H and n is 1.

In a particular embodiment of CA1, X is OH, n=1, and R¹-R⁷ are H. In a particular embodiment of CA1, X is a halogen. In a more particular embodiment, the halogen is selected from the group consisting of Cl or F. In an embodiment of CA1, X is Cl. In an embodiment of CA1, X is F. In an embodiment of CA1, X is not H.

In other specific embodiments, the invention includes compounds of formula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH or H and R² is selected from the group consisting of H, optionally substituted alkyl groups, optionally substituted alkoxy groups and optionally substituted phenyl groups. Of particular interest are those compounds in which the optional substitution is one or more halogens, including one or more fluorines or chlorines.

In other specific embodiments, the invention includes compounds of formula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH and R² is selected from the group consisting of H, optionally substituted alkyl groups, optionally substituted alkoxy groups and optionally substituted phenyl groups. Of particular interest are those compounds in which the optional substitution is one or more halogens, including one or more fluorines or chlorines.

In other specific embodiments, the invention includes compounds of formula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH or H and R² is selected from the group consisting of optionally substituted alkyl groups, optionally substituted alkoxy groups and optionally substituted phenyl groups. Of particular interest are those compounds in which the optional substitution is one or more halogens, including one or more fluorines or chlorines.

In other specific embodiments, the invention includes compounds of formula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH and R² is selected from the group consisting of optionally substituted alkyl groups, optionally substituted alkoxy groups and optionally substituted phenyl groups. Of particular interest are those compounds in which the optional substitution is one or more halogens, including one or more fluorines or chlorines.

In other specific embodiments, the invention includes compounds of formula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH or H and R² is selected from the group consisting of H, alkyl groups, alkoxy groups and a phenyl group.

In other specific embodiments, the invention includes compounds of formula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH and R² is selected from the group consisting of H, alkyl groups, alkoxy groups and a phenyl group.

In other specific embodiments, the invention includes compounds of formula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH or H and R² is selected from the group consisting of H, a methyl group, an ethyl group, propyl groups, butyl groups, a methoxy group, an ethoxy group, propyloxy groups, butyloxy groups and a phenyl group.

In other specific embodiments, the invention includes compounds of formula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH and R² is selected from the group consisting of H, a methyl group, an ethyl group, propyl groups, butyl groups, a methoxy group, an ethoxy group, propyloxy groups, butyloxy groups and a phenyl group.

In other specific embodiments, the invention includes compounds of formula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH or H and R² is selected from the group consisting of H, a methyl group, a methoxy group, and a phenyl group.

In other specific embodiments, the invention includes compounds of formula CA1, wherein n is 1, all of R¹, R³-R⁷ are hydrogens, X is OH and R² is selected from the group consisting of H, a methyl group, a methoxy group, and a phenyl group.

In a specific embodiment, compounds 278, 297, 300 and 446; and pharmaceutically acceptable salts, and esters thereof; are useful for treatment of a bone resorption clinical disorder.

In a specific embodiment, compounds 278, 297, 300, 444, 445 and 446; and pharmaceutically acceptable salts, and esters thereof; are useful in treatment of protozoan diseases, useful for treatment of a bone resorption clinical disorder, and for immunotherapy.

In a specific embodiment, compounds, the des-hydroxy (where X is H) analogs of compounds 278, 297, 300, 444, 445 and 446; and pharmaceutically acceptable salts, and esters thereof; are useful in the treatment of a bone resorption clinical disorder.

Compounds of this invention and compounds useful in the methods of this invention include those of the above formulas and pharmaceutically-acceptable salts and esters of those compounds. Salts include any salts derived from the acids of the formulas herein which acceptable for use in human or veterinary applications. The term esters refers to hydrolyzable esters of diphosphonate compounds of the formulas herein. Salts and esters of the compounds of the formulas herein are those which have the same therapeutic or pharmaceutical (human or veterinary) properties as the diphosphonate compounds of the formulas herein. Various combinations of salts are possible, with each phosphonate carrying a 2-, 1- or neutral charge. In principle there are multiple charge states possible, for example 9 charge states, for certain bisphosphonates of this invention.

In an embodiment, the invention provides a compound selected from the group consisting of 278, 297, 300, 335, 344, 359, 364, 398, 443-447, 449-452, 455-457, 459-462, 470-481, 483-485, ZZ1, 502, 511, 513, 520, 521, 523-526, 529-534, 542, 556, 577-579, 582, 583, 586, 588, 590, 591, 595, 597-605, 607, 610, 612, and 613; and for each respective said compound, a pharmaceutically acceptable salt or ester thereof.

In an embodiment, the invention provides a therapeutic composition comprising one or more compounds selected from the group consisting of 278, 297, 300, 335, 344, 359, 364, 398, 443-447, 449-452, 455-457, 459-462, 470-481, 483-485, ZZ1; and for each numbered compound a pharmaceutically acceptable salt or ester thereof; wherein the compounds are present in the composition in an amount or in a combined amount effective for obtaining the desired therapeutic benefit. The therapeutic compositions of this invention optionally further comprise a pharmaceutically acceptable carrier as known in the art.

In a specific embodiment, the invention includes compounds of the above formula CA1 where n=1, R¹ and R³-R⁷ are hydrogens, X═OH, and R²═H, optionally substituted alkyl, optionally substituted alkoxy, and optionally substituted phenyl. In a more specific embodiment, the invention includes compounds where n=1, R¹ and R³-R⁷═H, X═OH, and R²═H, alkyl, alkoxy, and phenyl. In a further specific embodiment, the invention includes compounds where n=1, R¹ and R³-R⁷═H, X═OH, and R²═H, methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, or phenyl.

In an embodiment, the invention provides various methods relating to the treatment of clinical disease. In an embodiment, the invention provides a method of treating a bone resorption disorder comprising administering to a patient in need a composition comprising a compound of the invention.

In an embodiment, the invention provides a method of treating a cancer disorder comprising administering to a patient in need a composition comprising a compound of the invention. In a specific embodiment, the cancer is breast cancer. In a specific embodiment, the breast cancer involves an actual or potential bone metastatic condition. In a specific embodiment, the invention provides a method of treating myeloma, lymphoma, prostate cancer, an epidermoid cancer, or orthotopic tumors.

In an embodiment, the invention provides compounds and methods for use in a combination therapy in the treatment of cancer. In a specific embodiment, a combination therapy utilizes a bisphosphonate compound of the invention and a different chemotherapeutic agent which can optionally be a distinct other bisphosphonate compound. In a particular embodiment the different chemotherapeutic agent is alendronate, zoledronate, risedronate, pamidronate, fas ligand (FasL), mevastatin, dexamethasone, paclitaxel, epirubicin, docetaxel, imatinib mesylate, tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), uracil-tegafur, gemcitabine, melphalan, doxorubicin, vincristine, or R115777 farnesyl transferase inhibitor (FTI) (Zarnestra®). In a particular embodiment, the combination of the bisphosphonate compound of the invention and the different chemotherapeutic agent has a synergistic effect. In another particular embodiment the combination has an additive effect.

In an embodiment, the invention provides a method of treating an infectious disease comprising administering to a patient in need a composition comprising a compound of the invention. In a specific embodiment, the infectious disease relates to an agent selected from the group consisting of: a virus, a bacterium, a fungus, and a protozoan parasite. In a specific embodiment, the virus is a retrovirus. In a more specific embodiment, the retrovirus is human immunodeficiency virus (HIV). In an embodiment, the protozoan parasite is Leishmania major. In an embodiment, the protozoan parasite is selected from the group consisting of: Leishmania, Toxoplasma, Cryptosporidium, Plasmodium, and Trypanosoma. In an embodiment, the infectious disease is selected from the group consisting of leishmaniasis, toxoplasmosis, cryptosporidiosis, sleeping sickness, and malaria.

In an embodiment, the invention provides a method of immunotherapy comprising administering to a patient in need a composition comprising a compound of the invention. In a specific embodiment, the method stimulates T cells in the patient. In a more specific embodiment, the method stimulates gamma delta T cells.

In an embodiment, the invention provides a method of screening a bisphosphonate test compound for a potential therapeutic activity, comprising: providing said bisphosphonate test compound, measuring a performance attribute of said test compound in at least three assays selected from the group consisting of: a Leishmania major farnesyl diphosphate synthase (FPPS) assay, a Dictyostelium discoideum assay, a T cell activation assay, and a bone resorption assay, analyzing said performance attribute; and selecting said bisphosphonate test compound based on said attribute; thereby screening said bisphosphonate test compound. In a specific embodiment, the method further comprises providing a reference compound and comparing a performance attribute of said reference compound with said performance attribute of said test compound.

In an embodiment, the invention provides a method of synthesizing a bisphosphonate compound of the invention, for example formula CA1, comprising: syntheses as shown and described herein, e.g. in schemes, FIG. 2, etc.; and as further would be understood in the art.

In an embodiment, the invention provides a method of treating bone pain comprising administering to a patient in need a compound of the invention. In a particular embodiment, the treatment of bone pain is in the context of a bone disease. In a particular embodiment, the treatment of bone pain is in the context of a patient with a metastatic cancer. In a particular embodiment, the metastatic cancer has spread to a bone location or originated in a bone location. For example, the treatment of bone pain can be achieved in a breast cancer patient wherein a metastatic breast cancer can or has spread to a bone location.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates compounds C1-C6, including pamidronate (Aredia®) C1; alendronate (Fosamax®) C2; risedronate (Actonel®) C3; zoledronate (Zometa®) C4; C5; and C6 shown in their zwitterionic forms.

FIG. 2 illustrates synthetic routes in the preparation of compounds.

FIG. 3 illustrates structures of compounds.

FIG. 4 illustrates structures of selected compounds.

FIG. 5 illustrates structures of selected compounds.

FIG. 6 illustrates cellular pathways of isoprenoid biosynthesis.

FIG. 7 illustrates the structures of pyridinium-1-yl bisphosphonates, compounds C5 and C7-C18.

FIG. 8 illustrates correlations: A, between FPPS inhibition and D. discoideum growth inhibition; and B, between FPPS inhibition and γδ T cell activation (as determined by TNF-α release). The R² and p values are R²=0.65 and p<0.0001 in A and R²=0.68 and p<0.0001 in B.

FIG. 9 illustrates a structure of a bisphosphonate compound.

DETAILED DESCRIPTION OF THE INVENTION

The invention may be further understood by the following non-limiting examples.

Example 1 Bisphosphonate Compounds

We report the design, synthesis and testing of a series of novel bisphosphonates. The most potent molecules have high activity and can represent useful compositions for a variety of applications such as in bone resorption disorders, parasitic diseases, immunomodulation, and cancer.

Our efforts led to the prediction of the importance of the presence of a positive charge at a relatively localized position in the bisphosphonate side chain. This can be related to the position of the positive charge expected in the pyridinium and imidazolium forms of compounds C3 and C4, shown above.

We explored the possibility that the pyridinium-1-yl species, compound C5, might have useful activity. We synthesized C5 and a series of derivatives (compounds C7-C18, FIG. 7), using the following general scheme, Scheme 1.

In the first step, we used (where necessary) coupling reactions of arylmetallic compounds with bromopyridines, catalyzed by Pd(PPh₃)₄ (ref. 32) or NiCl₂(PPh₃)₂ (ref. 33) to produce substituted pyridines. The substituted pyridines were then alkylated by using bromoacetic acid (ref. 34) and the resulting pyridinium-1-yl acetic acids were converted to the corresponding bisphosphonates by using H₃PO₃/POCl₃ (ref. 35).

We then investigated the activity of each compound in inhibiting the FPPS from L. major, in Dictyostelium discoideum growth inhibition and in γδ T cell activation. We used the FPPS from L. major since this enzyme is the putative bisphosphonate target in several trypanosomatid species. We used D. discoideum to test for cell growth inhibition, since this organism has been useful in the context of the development of bone resorption drugs (ref. 36). To determine the stimulatory activity of compounds C5 and C7-C18 for Vγ2Vδ2 T cells, we used the TNF-α release assay (ref. 30).

In the L. major FPPS inhibition assay, C5 was found to have a Ki of 18 nM (Table 6) and was thus slightly less active than the most potent commercially available bisphosphonates, zoledronate (4, Ki=11 nM in this assay) and risedronate (3, Ki=17 nM in this assay) (Table 6). In order to try to enhance activity, we next investigated the desirability of placing substituents at the meta position. We thus prepared compounds C7-C9 (FIG. 7) and tested them in the FPPS assay. The meta-methyl analog of C5 (C7) was not as active as C5 (Ki=38 nM versus 18 nM), but substitution with a meta-phenyl group, giving C8, resulted in a very potent species, having a Ki=9 nM, slightly more active than zoledronate, 4 (Ki=11 nM). The para-phenoxy derivative of C5 (C9) was found to be less active (Ki=75 nM), possibly due to unfavorable electrostatic interactions of the OH group in the FPPS active site.

TABLE 1 Activities of bisphosphonates as L. major FPPS inhibitors, D. discoideum cell growth inhibitors and gammadelta T cell simulators.* gammadelta D. T cell L. major discoideum stimulation, Compound FPPS Ki IC₅₀ EC₅₀ Compound Alias (nM) (μM) (μM) 1 190 167 940 (pamidronate)^(a,b) 2 95 32 52 (alendronate)^(a,b) 3 17 2.8 6.2 (risedronate)^(a,b) 4 11 1.9 7.3 (zoledronate)^(a,b) 5 278 18 2.1 5.1 6 23 1.6 15 (incadronate)^(a,b) 7 297 38 1.5 4.6 8 300 9 2.8 3.7 9 359 75 2.9 24 10 335 160 8.5 430 11 344 70 9.9 140 12 364 950 2.3 >1000 13 398 80 20 41 14 447 380 72 53 15 444 20 5.6 5.1 16 445 20 2.9 5.2 17 446 30 6 4.6 18 443 110 12 230 ^(a) L. mayor FPPS inhibition data from Ref. 17 ^(b)Gammadelta T cell stimulation data from Ref. 30 *Note that compound designations here are referred to by numbers and may optionally be designated with a preceding “C” yielding, e.g., C1, C2, etc.

Since the phenylpyridinium species, C8, displayed good activity, we next synthesized C10 and C11. Both of these compounds contain a methylene linker between the two aromatic groups and it seemed possible that they might better mimic the putative geranyl diphosphate reactive intermediate (C19):

but each of these compounds was approximately ten-fold less active than C8, with Ki values in the 70-160 nM range, Table 1. We also prepared the biphenylpyridinium compound, C12, since the added hydrophobicity of C8 appeared encouraging, but C12 proved to be relatively inactive, having a Ki of 950 nM. The isoquinoline and quinoline species, C13 and C14, had modest activity (80 and 380 nM, respectively, for C13 and C14), but the meta- ethyl (C15), butyl (C16), methoxy (C17) and para-benzyl (C18) pyridinium species were generally more active (20, 20, 30 and 110 nM, respectively), although they were less active than compounds C3-C6, C8. See Table 1.

We next investigated D. discoideum growth inhibition by compounds 1-18 (Table 1; alternatively referred to as C1-C18). The most active compound found was the meta-methyl pyridinium compound, C7, which had an IC₅₀ of 1.5 μM, followed by incadronate (C6, IC₅₀=1.6 μM) and zoledronate (C4, IC₅₀=1.9 μM). The unsubstituted pyridinium bisphosphonate, C5, was slightly more active (IC₅₀=2.1 μM) than was risedronate (C3, IC₅₀=2.8 μM). As with the FPPS inhibition results, the benzylpyridinium bisphosphonates (C10,C11,C18) were less active than the pyridinium and phenylpyridinium species (C5, C7-9). Surprisingly, C12 showed high activity, due perhaps to the possibility of an additional target in D. discoideum or the possibility of structural differences between L. major and D. discoideum FPPS enzymes. With the exception of C12, the activity results for the 17 bisphosphonates are highly correlated (R²=0.65, p<0.0001), as shown in FIG. 8A.

Next, we investigated the ability of C5 and compounds C7-C18 to stimulate gammadelta T cells, using the TNF-α release assay (ref. 30). The most active compound was found to be C8 (EC₅₀=3.7 μM), followed by C7 and C17 (EC₅₀=4.6 μM), with these compounds having more activity than risedronate (C3, EC₅₀=6.2 μM) (ref. 30) or zoledronate (C4, EC₅₀=7.3 μM) (ref. 30) in this TNF-α release assay. Addition of the para hydroxyl group (8→9) again reduced activity (Table 1), and again all three methylene bridged compounds (C10,C11,C18) had poor activity. The activity results for FPPS inhibition were found to be highly correlated with gammadelta T cell TNF-α release results (R²=0.68, p<0.0001), as shown in FIG. 8B, suggesting the likely importance of FPPS inhibition in gammadelta T cell activation (refs. 29,30).

We also explored the idea that electron withdrawing substituents on the ring could improve activity in bone resorption. To test this, compound 461 was prepared and tested, and indeed this species was found to be very potent in bone resorption. See Table 4.

Without wishing to be bound by a particular theory, these results may confirm the importance of a positive charge at the N-1 position. This suggests that the further development of this class of compounds can continue to be of interest in the context of the chemotherapy of infectious diseases, bone resorption, cancer, bone pain, and in immunotherapy.

Example 2 Structures of Bisphosphonate Compounds

Particular bisphosphonate compounds were synthesized. See Table 2 and the following figures: FIG. 3, FIG. 4, and FIG. 5. Note that in Table 2, the first column with the heading “Item” is not intended to refer to a compound designation, whereas the second column does refer to compound designations.

TABLE 2 Summary list of compounds with corresponding structures illustrated in FIGS. 3–5. Compound Item Designation 1 278 2 297 3 300 4 335 5 344 6 359 7 364 8 398 9 443 10 444 11 445 12 446 13 447 14 449 15 450 16 451 17 452 18 455 19 456 20 457 21 459 22 460 23 461 24 462 25 470 26 471 27 472 28 473 29 474 30 475 31 476 32 477 33 478 34 479 35 480 36 481 37 483 38 484 39 485 40 ZZ1 41 502 42 511 43 513 44 520 45 521 46 523 47 524 48 525 49 526 50 529 51 530 52 531 53 532 54 533 55 534 56 542 57 556 58 577 59 578 60 579 61 582 62 583 63 586 64 588 65 590 66 591 67 595 68 597 69 598 70 599 71 600 72 601 73 602 74 603 75 604 76 605 77 607 78 610 79 612 80 613

Example 3 Activity of Bisphosphonate Compounds in T Cell Stimulation an Applications in Immunotherapy

Additional compounds were tested for the ability to stimulate gammadelta T cells. Results are shown in Table 3.

TABLE 3 gammadelta T cell stimulation results for selected bisphosphonate compounds. Compound EC₅₀ (μM) 2 4.7 2 3.5 278 4.8 297 4.9 300 2.1 335 63.4 398 40.6 442 27.5 443 186.6 444 2.8 445 2.5 461 2.7 470 3.3 472 2.1 473 20.1 474 2.4 475 2.8 476 2.0 477 1.8 480 68.7 481 83.4 482 12.5 483 2.9 484 4.6

Example 4 Exploration of Activity of Bisphosphonate Compounds and Strategic Design of Compounds

We explored the hypothether analogs of pyrophosphate could block pyrophosphase enzymes and/or inhibit cellular growth or function. We used certain bisphosphonate compounds that are currently applied in bone resorption therapy. The compounds included pamidronate (Aredia®, Novartis), alendronate (Fosamax®, Merck), and risedronate (Actonel®, Procter & Gamble). The compounds did not appear necessarily to inhibit pyrophosphatases but did inhibit parasite cell growth. Without wishing to be bound by a particular theory, the compounds appeared to act as inhibitors of isoprenoid biosynthesis (FIG. 6).

The FPP synthase inhibitor pamidronate was observed to be effective in treating cutaneous leishmaniasis in mice. The average lesion size in treated mice was reduced during a time period of several weeks and in a dose-dependent manner relative to treated mice.

In an embodiment, a compound of the invention inhibits deoxyxylulose-5-phosphate reductoisomerase (DXR), an enzyme involved in isoprenoid biosynthesis. In a particular embodiment, a compound is able to affect Plasmodium in vitro or in vivo.

In an embodiment of the invention, a compound inhibits the mevalonate pathway. In an embodiment of the invention, a compound interacts with IPP isomerase (next to FPP synthase in the isoprene biosynthesis pathway) and activates gammadelta T cells.

Example 5 Activity of Bisphosphonate Compounds in Bone Resorption

Compounds are tested in a bone resorption assay: ⁴⁵Ca²⁺ release from 17-day old fetal mouse metatarsals (ref. 37). Results of IC₅₀ values for test compounds are observed and optionally compared to those for reference compounds such as risedronate (C3), alendronate (C2), and pamidronate (C1) and/or other bisphosphonate compounds known in the art. In a preferred embodiment, bisphosphonates of the invention such as the pyridinium-1-yl bisphosphonates are comparable to or more active in the bone resorption assay or in treatment of a bone resorption clinical disorder than one or more other reference bisphosphonates.

Example 6 Application of Bisphosphonate Compounds in the Treatment of Cancer

Compounds are tested for efficacy in reducing the occurrence, severity, or course of bone metastases in stage II/III breast cancer patients. A compound of the invention is found effective and administered to a patient in need of treatment. Treatment with a compound of the invention is effective in reducing the risk of bone metastasis and/or increasing the likelihood of survival, optionally in relation to treatment with a placebo. A compound is effective in enhancing a survival outcome in patients with more advanced disease. A compound administered to a cancer patient can simultaneously provide a benefit in the treatment of osteolysis and/or hypercalcemia while assisting in the prevention of bone metastasis and significantly increasing overall survival in breast cancer patients.

Compositions of the invention are applied in the treatment of skin metastases and mediastinal lymphomas. See Wilhelm et al., 2003.

Compositions of the invention are useful in the treatment of cancers such as lymphoma and myeloma and/or other forms of cancer. See Green J R, 2004, The Oncologist 9(supp 4):3-13; Forsea A-M et al., 2004, British Journal of Cancer 91:803-810.

Compositions of the invention are used in a combination therapy in the treatment of cancer. In a specific embodiment, a combination therapy utilizes a bisphosphonate compound of the invention and a different chemotherapeutic agent which can optionally be a distinct other bisphosphonate compound. See Caraglia M et al., 2004, Oncogene 23:6900-6913. See Salomo M et al., 2003, British Journal of Haematology 122:202-210.

Example 7 Application of Bisphosphonate Compounds in the Treatment of HIV Infection and AIDS

Many HIV drugs are suboptimally effective in partial relation to mutations of HIV-1 reverse transcriptase that confer resistance to a drug. For example, the effectiveness of azidothymidine (AZT; zidovudine, Retrovir), is believed to be so diminished. Bisphosphonate compounds of the invention are used in conjunction with AZT to provide an improved composition and therapy. Without wishing to be bound by a particular theory, a bisphosphonate compound inhibits AZT excision caused by ATP or PPi; the inhibition results in increased AZT activity in enzyme and cellular assays. A reversion of resistance phenotype is achieved by rendering an HIV-1 strain more sensitive to AZT activity.

Example 8 Additional Bisphosphonate Compounds

Further bisphosphonate compounds were synthesized. The structures are indicated in FIG. 9, FIG. 10, and FIG. 11. As noted in other Examples herein, the functional activity of certain of these compounds was assessed.

Example 9 Bisphosphonate Compounds have Activity in Several Contexts Relating to Bone Resorption and Immune Regulation

We have obtained data for selected bisphosphonate compounds in the contexts of D. discoideum assays, human FPPS assays, gammadelta T cell stimulation assays, and bone resorption assays. Several compounds showed substantial activity in one or more functional assays. Some toxicity testing was also performed. See Table 4 (in some instances, the same data depicted elsewhere herein, e.g., in Table 1, may be presented again to facilitate comparative analysis).

TABLE 4 (3) (5) gammadelta (4) Toxicity IC₅₀ (1) (2) T cell Bone (ug/mL - human D. discoideum human FPPS stimulation resorption nasopharyngeal Compound IC₅₀ (uM) IC₅₀ (uM) EC₅₀ IC₅₀ (uM) carcinoma cells) 278 2.6 1.6 5.1 0.67 300 297 2.8 NT 4.6 0.22 5.3 300 2.8 1.7 3.7 0.41 5.4 335 11 NT 430 NT NT 344 8.5 17 140 NT NT 359 2.9 NT 24 NT NT 364 2.3 37 >1000 NT NT 398 20 NT 41 NT NT 443 12 NT 230 NT NT 444 5.6 NT 5.1 NT NT 445 3 NT 5.2 NT NT 446 6 NT 4.6 0.37 NT 447 72 NT 53 NT NT 449 4 NT 12 NT NT 450 4.2 NT 6.8 NT NT 451 31 280 inactive NT NT 452 73 NT 250 NT NT 455 27 NT 25 NT NT 456 27 NT 19 NT NT 457 14 NT 1900 NT NT 459 27 NT 14 NT NT 460 14 NT 13 NT NT 461 3.7 NT 2.7 0.075 NT 462 2.7 NT 15 NT NT 470 3.5 NT NT NT NT 471 inactive NT inactive NT NT 472 2.5 NT NT NT NT 473 20 NT NT NT NT 474 4.4 NT NT NT NT 475 4.1 NT NT NT NT 476 2.3 NT NT NT NT 477 2.7 1.4 NT NT NT 478 760 NT inactive NT NT 479 6 NT 15 NT NT 480 3 NT NT NT NT 481 3.5 NT NT NT NT 483 2.5 NT NT NT NT 484 3.5 2.5 NT NT NT 485 58 NT 370 NT NT 502 100 NT inactive NT NT 511 4.1 2.5 14 NT NT 513 1500 NT 550 NT NT 520 6.3 NT 14 NT NT 521 570 NT 790 NT NT 523 4.6 NT 11 NT NT 524 2.4 NT 10 NT NT 525 8.4 NT 11 NT NT 526 2.1 1.2 8.6 NT NT 529 3.3 NT 9.4 NT NT 530 190 NT 6500 NT NT 531 20 NT inactive NT NT 532 4.1 NT 41 NT NT 533 NT NT 19 NT NT 534 no tested NT 16 NT NT 542 1.7 1.2 12 NT NT 556 inactive NT 2300 NT NT 577 4.1 NT 21 NT NT 578 4 NT 20 NT NT 579 6 NT 20 NT NT 582 3.5 1.6 NT NT NT 583 14 NT 50 NT NT 586 6 NT 90 NT NT 588 3.3 11 120 NT NT 590 9.1 23 300 NT NT 591 38 29 NT NT NT 595 49 2.7 210 NT NT 597 2.0 1.9 58 NT NT 598 1.7 NT 35 NT NT 599 1.7 NT 69 NT NT 600 11 NT 290 NT NT 601 2.4 NT 220 NT NT 602 2.04 NT 18 NT NT 603 NT NT 22 NT NT 604 NT NT 10 NT NT 605 NT NT 6 NT NT 607 NT NT NT NT NT 610 NT NT NT NT NT 612 NT NT NT NT NT 613 NT NT NT NT NT 614 NT NT NT NT NT 615 NT 5.2 NT NT NT NT, not tested.

Example 10 Activity of Bisphosphonate Compounds Against Trypanosoma and Leishmania Parasites

We have obtained data for selected bisphosphonate compounds against parasites including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. Several compounds showed substantial activity in one or more functional assays. See Table 5 (in some instances, the same data depicted elsewhere herein, e.g., in Table 1, may be presented again to facilitate comparative analysis).

TABLE 5 (9) (6) (7) T. brucei soluble T. brucei L. major (8) vacuolar FPPS FPPS T. cruzi pyrophosphatase Compound IC₅₀ (uM) Ki (nM) IC₅₀ (ug/mL) IC₅₀ (uM) 278 0.83 18 3.07 NT 297 NT 38 NT NT 300 0.58 9 NT 35 335 NT 160 NT 70 344 NT 70 NT 78 359 NT 75 NT NT 364 NT 950 NT NT 398 NT 80 NT NT 443 NT 110 NT NT 444 NT 20 NT NT 445 NT 20 NT NT 446 NT 30 NT NT 447 NT 380 NT NT 449 NT NT NT NT 450 NT NT NT NT 451 NT NT NT NT 452 NT NT NT NT 455 NT NT NT NT 456 NT NT NT NT 457 NT NT NT NT 459 NT NT NT NT 460 NT NT NT NT 461 0.54 50 NT NT 462 NT NT NT NT 470 0.79 NT NT NT 471 NT NT NT NT 472 NT NT NT NT 473 NT NT NT NT 474 NT NT NT NT 475 NT NT NT NT 476 NT NT NT NT 477 0.89 NT NT NT 478 NT NT NT NT 479 NT NT NT NT 480 NT NT NT NT 481 NT NT NT NT 483 0.43 NT NT NT 484 1.1 NT NT NT 485 NT NT NT NT 502 NT NT NT NT 511 0.76 NT NT NT 513 NT NT NT NT 520 0.65 NT NT NT 521 NT NT NT NT 523 NT NT NT NT 524 0.26 NT NT NT 525 NT NT NT NT 526 0.54 NT NT NT 529 NT NT NT NT 530 NT NT NT NT 531 NT NT NT NT 532 NT NT NT NT 533 NT NT NT NT 534 NT NT NT NT 542 0.39 NT NT NT 556 NT NT NT NT 577 NT NT NT NT 578 NT NT NT NT 579 NT NT NT NT 582 NT NT NT NT 583 4.6 NT NT NT 586 NT NT NT NT 588 NT NT NT NT 590 NT NT NT NT 591 NT NT NT NT 595 NT NT NT NT 597 NT NT NT NT 598 0.59 NT NT NT 599 0.54 NT NT NT 600 NT NT NT NT 601 2.1 NT NT NT 602 1 NT NT NT 603 0.71 NT NT NT 604 0.57 NT NT NT 605 0.29 NT NT NT 607 NT NT NT NT 610 NT NT NT NT 612 NT NT NT NT 613 NT NT NT NT 614 NT NT NT NT 615 NT NT NT NT NT, not tested.

Statements Regarding Incorporation by Reference and Variations

All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).

When a group of substituents is disclosed herein, it is understood that all individual members of those groups and all subgroups, including any isomers and enantiomers of the group members, and classes of compounds that can be formed using the substituents are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. When a compound is described herein such that a particular isomer or enantiomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomer and enantiomer of the compound described individually or in any combination. When an atom is described herein, including in a composition, any isotope of such atom is intended to be included. Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently. Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure.

All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art, in some cases as of their filing date, and it is intended that this information can be employed herein, if needed, to exclude (for example, to disclaim) specific embodiments that are in the prior art. For example, when a compound is claimed, it should be understood that compounds known in the prior art, including certain compounds disclosed in the references disclosed herein (particularly in referenced patent documents), are not intended to be included in the claim.

Where the terms “comprise”, “comprises”, “comprised”, or “comprising” are used herein, they are to be interpreted as specifying the presence of the stated features, integers, steps, or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component, or group thereof.

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention. It will be apparent to one of ordinary skill in the art that methods, devices, device elements, materials, procedures and techniques other than those specifically described herein can be applied to the practice of the invention as broadly disclosed herein without resort to undue experimentation. All art-known functional equivalents of methods, devices, device elements, materials, procedures and techniques described herein are intended to be encompassed by this invention. Whenever a range is disclosed, all subranges and individual values are intended to be encompassed. This invention is not to be limited by the embodiments disclosed, including any shown in the drawings or exemplified in the specification, which are given by way of example or illustration and not of limitation.

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1. A compound having the formula CA1:

or a pharmaceutically acceptable salt thereof; wherein: X is —OH, or a halogen; n is 1, 2, or 3; R¹-R⁵, independently of one another are selected from the group consisting of a hydrogen, a halogen, a —CN, —OR, —COOR, —OCOOR, —COR, —CON(R)₂, —OCON(R)₂, —N(R)₂, —NO₂, —SR, —SO₂R, —SO₂N(R)₂or —SOR group, an optionally substituted alkyl group, an optionally substituted alkenyl group, and an optionally substituted aryl group, where each R, is selected from H, an optionally substituted alkyl group and an optionally substituted aryl group, an optionally substituted acyl group; and R⁶ and R⁷, independently of each other are selected from the group consisting of a hydrogen, a halogen, a —N(R)₂, or —SR group, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkoxy group, and an optionally substituted aryl group, where each R, is selected from H, an optionally substituted alkyl group and an optionally substituted aryl group; and wherein R⁶ and R⁷ can together form a ring which may contain one or more double bonds.
 2. The compound of claim 1 wherein X is OH, n=1, and R¹-R⁷ are H.
 3. The compound of claim 1 wherein X is the halogen and the halogen is selected from the group consisting of Cl or F.
 4. The compound of claim 1 wherein X is Cl.
 5. The compound of claim 1 wherein X is F.
 6. A compound having the formula CA1:

or a pharmaceutically acceptable salt thereof; wherein: X is —OH; n is 1, 2, or 3; R¹-R⁵, independently of one another are selected from the group consisting of a hydrogen, a halogen, a —CN, —OR, —COOR, —OCOOR, —COR, —CON(R)₂, —OCON(R)₂, —N(R)₂, —NO₂, —SR, —SO₂R, —SO₂N(R)₂or —SOR group, an optionally substituted alkyl group, an optionally substituted alkenyl group, and an optionally substituted aryl group, where each R, is selected from H, an optionally substituted alkyl group and an optionally substituted aryl group, an optionally substituted acyl group; and R⁶ and R⁷, independently of each other are selected from the group consisting of a hydrogen, a halogen, a —N(R)₂, or —SR group, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkoxy group, and an optionally substituted aryl group, where each R, is selected from H, an optionally substituted alkyl group and an optionally substituted aryl group; and wherein R⁶ and R⁷ can together form a ring which may contain one or more double bonds.
 7. A compound of selected from the group of compounds consisting of compounds 278, 297, 300, 335, 344, 359, 364, 398, 443-447, 449-452, 455-457, 459-462, 471, 478, 479, 484, 513, 520, 524, 534, 582, 583, 590, 591, 595, 600, 607, 612, 613, ZZ1 and pharmaceutically acceptable salts thereof, the chemical formulas of which compounds are:


8. A compound of claim 1 selected from the group of compounds consisting of compounds 278, 297, 300, 335, 344, 359, 364, 444, 445, 446 and pharmaceutically acceptable salts thereof, the chemical formulas of which compounds are:


9. A compound of claim 1 selected from the group of compounds consisting of compounds 278, 297, 300, 444, 445, 461, 484 and pharmaceutically acceptable salts thereof, the chemical formulas of which compounds are:


10. A compound selected from the group of compounds consisting of compounds 472, 476, 477, 480, 481, 483, 485, 502, 511, 521, 523, 526, 529-533, 542, 556, 577-579, 586, 588, 597-599, 601-605, 610 and pharmaceutically acceptable salts thereof, the chemical formulas of which compounds are:


11. A compound of claim 10 selected from the group of compounds consisting of compounds 472, 476, 477, 483, and pharmaceutically acceptable salts thereof, the chemical formulas of which compounds are:


12. A compound of claim 10 selected from the group consisting of the compounds 476, 577-579, 586, 588, 597-599, 601-605, 610, and pharmaceutically acceptable salts thereof, the chemical formulas of which compounds are: 